I propose to develop the potential of the SV40 genetic system so that it may become a useful probe into eucaryotic recombination mechanism. As a basic for those studies, I will carry out fine-structure genetic analysis of SV40, using a novel two-step approach that relies on in vitro construction of a presumptive intermediate in SV40 recombination in order to bypass the apparent rate-limiting step in vivo. This combined in vitro-in vivo approach to recombination should permit fine-structure genetic analysis of SV40 that rivals any procaryotic system; the approach lends itself to 3-factor as well as 2-factor crosses, has a resolving power of 0.01% recombination per nucleotide, should not require a mapping function to relate percent recombination to genetic distance, and is expected to yield intermutation distances directly in terms of nucleotides. Within this proposal I outline a simple in vitro method for constructing the multiple mutants of SV40 that are necessary for three-factor mapping. I also indicate how three-factor crosses can be used to evaluate the importance of branch migration and heteroduplex formation in recombination. Finally, I proposed to screen cells that are defective in repair of UV damage for any that are mismatch-repair and/or recombination deficient. Taken together, these studies constitute the beginnings of a molecular characterization of recombination in cultured mammalian cells.